Receptors are the molecules that inform the inside of a cell what is going on outside. There are three critical steps to the function of a cell surface receptor. The first is ligand binding, the second is transmembrane signal transduction, the third is assembly or activation of an intracellular signal initiation complex. In this proposal we outline the use of a novel approach to define TLR receptor assembly. TLRs are now widely appreciated as detectors of a broad variety of ligands, exogenous as well as endogenous. Their activation initiates innate immune inflammatory responses and promotes adaptive immune responses as well. On the positive side, they are critical for detection of infection and on the negative, are involved in septic shock. Understanding their mechanisms of activation and intracellular signaling is important for understanding, and perhaps altering, the consequences of their activation. Alternate means of activating them could also have value, perhaps as novel adjuvants. Typical, and useful, approaches to understanding assembly of a signaling complex by the intracellular domain of a receptor have been varied; they include such approaches as the yeast two-hybrid system, coimmunoprecipitation, and site directed mutagenesis. To this group, using the context of TLRs, we propose to add protein fragment complementation. Selected fragments of many proteins can associate to produce functional bimolecular complexes. In one approach an enzyme activity is generated when inactive fragments of an enzyme are brought together in an orientation such that they can recombine and catalyze the formation of a detectable product. Given the catalytic nature of enzyme reactions, this approach can be remarkably sensitive. In another approach fragments of fluorescent proteins become fluorescent when appropriately brought together. The enzyme we propose to exploit is beta-Iactamase and the fluorescent protein we propose to exploit is yellow fluorescent protein (YFP). Thus we have two goals: To define novel protein-protein interactions in the TLR system using co-immunoprecipitation, beta- lactamase and YFP fragment complementation and to make alterations to the complementation systems to improve their utility for our purposes.